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Li P.,The Key Laboratory of Molecular Microbiology and Technology | Wang L.,Nankai University | Feng L.,Tianjin Key Laboratory of Microbial Functional Genomics
Journal of Bacteriology | Year: 2013

The cold-tolerant bacterium Pusillimonas sp. strain T7-7 is able to utilize diesel oils (C5 to C30 alkanes) as a sole carbon and energy source. In the present study, bioinformatics, proteomics, and real-time reverse transcriptase PCR approaches were used to identify the alkane hydroxylation system present in this bacterium. This system is composed of a Rieske-type monooxygenase, a ferredoxin, and an NADH-dependent reductase. The function of the monooxygenase, which consists of one large (46.711 kDa) and one small (15.355 kDa) subunit, was further studied using in vitro biochemical analysis and in vivo heterologous functional complementation tests. The purified large subunit of the monooxygenase was able to oxidize alkanes ranging from pentane (C5) to tetracosane (C24) using NADH as a cofactor, with greatest activity on the C15 substrate. The large subunit also showed activity on several alkane derivatives, including nitromethane and methane sulfonic acid, but it did not act on any aromatic hydrocarbons. The optimal reaction condition of the large subunit is pH 7.5 at 30°C. Fe2+ can enhance the activity of the enzyme evidently. This is the first time that an alkane monooxygenase system belonging to the Rieske non-heme iron oxygenase family has been identified in a bacterium. © 2013, American Society for Microbiology. Source


Wang W.,Nankai University | Wang W.,Tianjin Key Laboratory of Microbial Functional Genomics | Ma T.,Nankai University | Lian K.,Nankai University | And 4 more authors.
PLoS ONE | Year: 2013

Sulfur can be removed from benzothiophene (BT) by some bacteria without breaking carbon-carbon bonds. However, a clear mechanism for BT desulfurization and its genetic components have not been reported in literatures so far. In this study, we used comparative transcriptomics to study differential expression of genes in Gordonia terrae C-6 cultured with BT or sodium sulfate as the sole source of sulfur. We found that 135 genes were up-regulated with BT relative to sodium sulfate as the sole sulfur source. Many of these genes encode flavin-dependent monooxygenases, alkane sulfonate monooxygenases and desulfinase, which perform similar functions to those involved in the 4S pathway of dibenzothiophene (DBT) biodesulfurization. Three of the genes were found to be located in the same operon, designated bdsABC. Cell extracts of pET28a-bdsABC transfected E. coli Rosetta (DE3) converted BT to a phenolic compound, identified as o-hydroxystyrene. These results advance our understanding of enzymes involved in the BT biodesulfurization pathway. © 2013 Wang et al. Source


Liu B.,Nankai University | Liu B.,The Key Laboratory of Molecular Microbiology and Technology | Knirel Y.A.,RAS N. D. Zelinsky Institute of Organic Chemistry | Feng L.,Nankai University | And 9 more authors.
FEMS Microbiology Reviews | Year: 2014

This review covers the structures and genetics of the 46 O antigens of Salmonella, a major pathogen of humans and domestic animals. The variation in structures underpins the serological specificity of the 46 recognized serogroups. The O antigen is important for the full function and virulence of many bacteria, and the considerable diversity of O antigens can confer selective advantage. Salmonella O antigens can be divided into two major groups: those which have N-acetylglucosamine (GlcNAc) or N-acetylgalactosamine (GalNAc) and those which have galactose (Gal) as the first sugar in the O unit. In recent years, we have determined 21 chemical structures and sequenced 28 gene clusters for GlcNAc-/GalNAc-initiated O antigens, thus completing the structure and DNA sequence data for the 46 Salmonella O antigens. The structures and gene clusters of the GlcNAc-/GalNAc-initiated O antigens were found to be highly diverse, and 24 of them were found to be identical or closely related to Escherichia coli O antigens. Sequence comparisons indicate that all or most of the shared gene clusters were probably present in the common ancestor, although alternative explanations are also possible. In contrast, the better-known eight Gal-initiated O antigens are closely related both in structures and gene cluster sequences. In this review, we systematically analyzed and summarized Salmonella O-antigen diversity including the chemical structures, gene cluster sequences, and evolutionary aspects. © 2013 Federation of European Microbiological Societies. Source


Zhu H.,Nankai University | Zhu H.,Key Laboratory of Molecular Microbiology and Technology | Wang Q.,Nankai University | Wang Q.,Key Laboratory of Molecular Microbiology and Technology | And 12 more authors.
Journal of Clinical Microbiology | Year: 2012

Neisseria meningitidis is a leading pathogen of epidemic bacterial meningitis and fulminant sepsis worldwide. Twelve different N. meningitidis serogroups have been identified to date based on antigenic differences in the capsular polysaccharide. However, more than 90% of human cases of N. meningitidis meningitis are the result of infection with just five serogroups, A, B, C, W135, and Y. Efficient methods of detection and genogrouping of N. meningitidis isolates are needed, therefore, in order to monitor prevalent serogroups as a means of disease control and prevention. The capsular gene complex regions have been sequenced from only seven out of the 12 serogroups. In this study, the capsular gene complexes of the remaining five serogroups were sequenced and analyzed. Primers were designed that were specific for N. meningitidis species and for the 12 individual serogroups, and a multiplex PCR assay using these specific primers was developed for N. meningitidis detection and genogrouping. The assay was tested using 15 reference strains covering all 12 serogroups, 143 clinical isolates, and 21 strains from closely related species or from species that cause meningitis. The assay could detect N. meningitidis serogroups and was shown to be specific, with a detection sensitivity of 1 ng of genomic DNA (equivalent to ∼4 × 10 5 genomes) or 3 × 10 5 CFU/ml in noncultured mock cerebrospinal fluid (CSF) specimens. This study, therefore, describes for the first time the development of a molecular protocol for the detection of all N. meningitidis serogroups. This multiplex PCR-based assay may have use for the clinical diagnosis and epidemiological surveillance of N. meningitidis. Copyright © 2012, American Society for Microbiology. All Rights Reserved. Source


Liu D.,Nankai University | Cai J.,Nankai University | Cai J.,Key Laboratory of Molecular Microbiology and Technology | Cai J.,Tianjin Key Laboratory of Microbial Functional Genomics | And 5 more authors.
Enzyme and Microbial Technology | Year: 2010

Chitinase A (ChiA) produced by Bacillus thuringiensis subsp. colmeri 15A3 (Bt. 15A3) was expressed in Escherichia coli XL-Blue. The ChiA was purified using Sephadex G-200 and its molecular mass was estimated to be 36 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Detection of chitinase activity on SDS-PAGE after protein renaturation indicated that the molecular mass of the protein band expressing chitinase activity was approximately 72 kDa. This suggests that the dimeric form of ChiA is the enzymatically active form when glycol chitin is used as a substrate. ChiA has optimal activity at 50 °C and retains most of its activity between 20 and 60 °C. The optimum pH for ChiA activity is pH 5.0, and the enzyme is active between pH 4.0 and 8.0. The enzyme activity was significantly inhibited by Ag+ and Zn2+. ChiA significantly inhibited the spore germination of four species of fungi. The median inhibitory concentrations (IC50) of ChiA on the spore germination of Penicillium glaucum and Sclerotinia fuckelian were 11.27 and 10.57 μg/ml, respectively. In surface contamination bioassays, the crude ChiA protein (12.6 mU) reduced the LC50 (50% lethal concentration) of the crystal protein of Bt. 15A3 against the larvae of Spodoptera exigua and Helicoverpa armigera. © 2009 Elsevier Inc. All rights reserved. Source

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